The adrenal glands, ovaries, testes, and placenta produce steroid intermediates, which are hydroxylated at position 21 (by 21-hydroxylase) and position 11 (by 11-hydroxylase) to produce cortisol. Deficiency of either 21-hydroxylase or 11-hydroxylase results in decreased cortisol synthesis and loss of feedback inhibition of adrenocorticotropic hormone (ACTH) secretion. The consequent increased pituitary release of ACTH drives increased production of steroid intermediates.

The steroid intermediates are oxidized at position 3 (by 3-beta-hydroxy dehydrogenase [3-beta-HSD]). The 3-beta-HSD enzyme allows formation of 17-hydroxyprogesterone (17-OHPG) from 17-hydroxypregnenolone and progesterone from pregnenolone. When 3-beta-HSD is deficient, cortisol is decreased, 17-hydroxypregnenolone and pregnenolone levels may increase, and 17-OHPG and progesterone levels, respectively, are low. Dehydroepiandrosterone is also converted to androstenedione by 3-beta-HSD, and may be elevated in patients affected with 3-beta-HSD deficiency.

The best screening test for CAH, most often caused by either 21- or 11-hydroxylase deficiency, is the analysis of 17-hydroxyprogesterone (along with cortisol and androstenedione). CAH21 / Congenital Adrenal Hyperplasia (CAH) Profile for 21-Hydoxylase Deficiency allows the simultaneous determination of these 3 analytes. Alternately, these tests may be ordered individually: OHPG / 17-Hydroxyprogesterone, Serum; CINP / Cortisol, Serum, LC-MS/MS; and ANST / Androstenedione, Serum.

If both 21- and 11-hydroxylase deficiency have been ruled out, analysis of 17-hydroxypregnenolone and pregnenolone may be used to confirm the diagnosis of 3-beta-HSD or 17-alpha-hydroxylase deficiency.

Diagnosis and differential diagnosis of congenital adrenal hyperplasia (CAH) always require the measurement of several steroids. Patients with CAH due to steroid 21-hydroxylase gene (CYP21A2) mutations usually have very high levels of androstenedione, often 5-fold to 10-fold elevations. 17-OHPG levels are usually even higher, while cortisol levels are low or undetectable. All 3 analytes should be tested.

For the HSD3B2 mutation, cortisol, 17-OHPG and progesterone levels will be will be decreased; 17-hydroxypregnenolone and pregnenolone and DHEA levels will be increased.

In the much less common CYP11A1 mutation, androstenedione levels are elevated to a similar extent as seen in CYP21A2 mutation, and cortisol also is low, but 17-OHPG is only mildly, if at all, elevated.

In the also very rare 17-hydroxylase deficiency, androstenedione, all other androgen-precursors (17-alpha-hydroxypregnenolone, 17-OHPG, dehydroepiandrosterone sulfate), androgens (testosterone, estrone, estradiol), and cortisol are low, while production of mineral corticoid and its precursors (in particular pregnenolone, 11-dexycorticosterone, corticosterone, and 18-hydroxycorticosterone) are increased.